Indirect ELISA Protocol

  1. Coat antigen on the ELISA plates, diluted in coating buffer, for 2 hours at RT.
  2. Wash off any unbound antigen from the wells.
  3. Block the wells using 3-5% BSA prepared in 1x PBS.
  4. Wash off excess blocking.
  5. Incubate with primary antibodies diluted in blocking buffer for 2 hours at RT or overnight in cold conditions at a dilution recommended by the manufacturer
  6. Wash off unbound primary antibodies.
  7. Add secondary antibodies diluted in blocking buffer at a dilution recommended by the manufacturer. Incubate at RT for 1 hour.
  8. Wash off excess secondary antibody.
  9. Develop the plate using the chromogenic substrate (TMB substrate when using HRP-labelled antibodies, pNPP for ALP labelled). Most substrates take around 10-30 minutes of reaction time.
  10. Stop the enzymatic reaction using a stop solution (0.1N H2SO4). 50-100ul of stop solution is recommended per well.
  11. Read the plate in a plate reader at specific wavelengths.

Note:

  • Caution must be exercised when selecting a secondary antibody. The tagged enzyme, the host it is grown in, and the organism it is raised against. If the antibody denotes ‘goat anti-rabbit’, the secondary antibody is raised in a goat but is against the rabbit. Similarly, if the label says goat anti-rabbit IgG, then the secondary antibody will only bind to Fc region of IgG antibodies in rabbit. In fact, the subclass IgG1 and IgG2 can be distinguished as conjugates of these subclasses are also available. Also the enzyme tagged with the secondary antibody should be noted as it determines the substrate to be used and directs the protocol. HRP-labelled secondary antibodies most commonly use TMB/H2O2 substrate.
  • Dilutions of both primary and secondary antibodies should always be made in the blocking reagent. This is to prevent non-specific binding of the antibody to the surface of the plate.
  • Incubation times can be minimized by incubating at 37°C for 30 min. Also, stationery conditions can be applied.
Comments Off on Indirect ELISA Protocol

Direct ELISA Protocol

  1. Coat antigen on the ELISA plates diluted in coating buffer for 2 hours at RT. Most protocols recommend using 100-300ng of purified protein per well. A maximum of 600-800ng can be added to each well in case the sample is more complex.
  2. Wash off any unbound antigen from the wells.
  3. Block the wells using 3-5% BSA prepared in 1x PBS, pH 7.5 and store the plate at 4°C overnight. Blocking can also be carried out at room temperature for 1 hour or 30 min at 37◦C.
  4. Wash off excess blocking. This step is optional as some antibodies do not need it.
  5. Incubate with primary antibodies diluted in blocking buffer for a minimum of 2 hours at RT or overnight in cold conditions (at a dilution recommended by the manufacturer).
  6. Wash off unbound primary antibodies.
  7. Develop the plate using the chromogenic substrate (TMB substrate when using HRP-labelled antibodies, pNPP for ALP labelled). Most substrates take around 10-30 minutes of reaction time.
  8. Stop the enzymatic reaction using a stop solution (0.1N H2SO4) of 50-100ul per well.
  9. Read the plate in a plate reader.

Note:

  • All washes are to be done using 1x PBST (0.01-0.1% Tween 20 added) three times for 5-10 minutes each at RT.
  • The plate can also read without stopping the reaction, but it should be done immediately after incubation. The stopping step allows for extra time for delayed plate reading.
  • The detection is through a single antibody species; hence, an enzyme-tagged monoclonal antibody should be used.
  • The output will be a reading of total antibodies against the target antigen. Classes of antibodies cannot be distinguished.
  • Incubations can be carried out at 37◦C and in stationery conditions.
Comments Off on Direct ELISA Protocol

Sandwich ELISA Protocol

  1. Coat the plate with the primary antibody in the coating buffer. This primary antibody is termed as 'capture antibody'.
  2. Wash off unbound capture
  3. Block plate with blocking buffer.
  4. Wash off the blocking buffer.
  5. Add the sample containing antigen, diluted in blocking buffer.
  6. Wash off unbound antigen.
  7. Add detection 'primary antibody' diluted in blocking buffer.
  8. Wash off unbound primary antibody.
  9. Add secondary antibodies diluted in blocking buffer at a dilution recommended by the manufacturer. Incubate at RT for 1 hour.
  10. Wash off excess secondary antibody.
  11. Develop the plate using the chromogenic substrate (TMB substrate when using HRP-labelled antibodies, pNPP for ALP labelled). Most substrates take around 10-30 minutes of reaction time.
  12. Stop the enzymatic reaction using a stop solution (0.1N H2SO4).50-100ul of stop solution is recommended per well.
  13. Read the plate in a plate reader.

Depending upon the use of an enzyme labelled primary or enzyme labelled secondary antibody, sandwich ELISAs can be termed as direct sandwich ELISA and an indirect sandwich ELISA. The above protocol discusses an indirect sandwich ELISA though a direct one also utilizes the same protocol with a change in the use of a labelled primary antibody.

Note:

  • Caution should be taken to choose antibodies for different epitopes. A simple way can be choosing a polyclonal antibody as a capture antibody and a monoclonal one for detection.
  • In a certain cases, when the detection antibody is unavailable, the capture antibody can be used as an alternative. However, this can compromise the accuracy of the method.
Comments Off on Sandwich ELISA Protocol

Glucosinolates estimation from de-oiled rapeseed cake using a spectrophotometer

Table of Contents
    Add a header to begin generating the table of contents

    Introduction

    Glucosinolates are plant secondary metabolites. Myrosinase is an enzyme that catalyzes the breakdown of glucosinolates. Both of them are usually present in different cells in the tissue, unless there is damage. The breakdown products differ depending on the environmental conditions.

    Applications

    1. Total glucosinolates estimation from various plants. This protocol aims at the mustard de-oiled cake.

    Advantages

    1. Less laborious than HPLC
    2. Simple extraction and estimation
    3. Relatively low cost

    Limitations

    1. This method has different affinities for different types of glucosinolates. This might lead to a skewed estimation of total Glucosinolate content. For example, sodium tetrachloropalladate reacts to all types of glucosinolates but has a higher affinity to indolyl glucosinolates.

    Reagents

    2 mM sodium tetrachloropalladate:

    58.8 mg Sodium tetrachloropalladate + 170 µl concentrated HCl +100 ml double distilled water.

    Procedure

    The protocol can be divided into extraction and estimation.

    Extraction of total Glucosinolates

    1. Homogenise 0.1 g defatted seed meal in a 2 ml vial with 80% methanol.
    2. Keep overnight at room temperature.
    3. Spin it at 3000 rpm for 4 min
    4. Collect supernatant.
    5. Make the volume to 2 ml with 80% methanol.

    Estimation of total Glucosinolates

    1. Take 100µL of the solution from the extraction step.
    2. Add 0.3 ml double distilled water
    3. 3 ml of 2 mM sodium tetrachloropalladate
    4. incubation at room temperature for 1 h
    5. The reaction mix turns brown. The intensity of the browning depends on the amount of glucosinolates.
    6. A blank was set following the same procedure without the extract.
    7. Find the O.D at 425nm use the O.D in the following formula to get the quantity of total Glucosinolates in µmol/g (Y).
    8. Y = 1.40 + 118.86 × A425
    9. Total glucosinolate content obtained by the predicted formula was comparable with the HPLC results.

    Caution

    • Using the right volumes is the key to the success of this protocol.
    • The extract preparation is very crucial, and therefore utmost care is needed before quantification, such as avoiding excessive shaking and vortexing of the extract. A brief centrifuge would suffice for good results.

    Reference

    1. A simple spectrophotometric method for estimating total glucosinolates in mustard de-oiled cake.
    2. Quantitative analysis of total glucosinolate content in concentrated extracts from double low rapeseed by the Pd-glucosinolate complex method.

    3. Small variation of glucosinolate composition in Japanese cultivars of radish (Raphanus sativus L.) requires simple quantitative analysis for breeding of glucosinolate component.

    Comments Off on Glucosinolates estimation from de-oiled rapeseed cake using a spectrophotometer
    Illustration showing steps of heat shock transformation of E.coli

    Heat shock transformation (E.coli) protocol

    Illustration showing steps of heat shock transformation of E.coli
    Table of Contents
      Add a header to begin generating the table of contents

      Introduction

      The heat shock method is one of the most used methods for transforming E.coli with the plasmid of interest.

      Applications

      • Propagation of plasmid DNA
      • Storage of plasmid DNA (in the form of glycerol stock)
      • Synthesis of proteins
      • To modify the metabolism of bacterial

      Reagents

      Instruments and other requirements

      • Incubator shaker.
      • Incubator.
      • Water bath or heat block.
      • Laminar flow hood.
      • Centrifuge.
      • Pipettes.
      • Sterile tips.
      • Spreader, and parafilm.

      Keep Following ready before starting the transformation

      1. Make sure laminar flow is available at the time of spreading (about 1.5-2 hr from the beginning of protocol).
      2. Make sure the incubator is available for the entire duration of the experiment and is set for 37°C.
      3. Keep the LB agar plates (with appropriate antibiotic) and media (or appropriate media) at room temperature or 37°C.
      4. Water bath or heat block at 42°C.

      Duration of the experiment

      the experiment takes nearly 2 hours to complete the transformation, 12-16 hours after transformation (spreading)for the colonies to appear on the plate.

      Procedure

      1. Take out the competent cells from -80◦C and keep them on ice for 5-10 mins.
      2. Add the ligation mix or plasmid to the competent cells and mix gently. Do not shake or vortex as it will inhibit transformation efficiency.
      3. Incubate the reaction mix on ice for 10-15 minutes.
      4. Give a heat shock to the cells by placing the reaction mix at 42°C for 30-90 seconds (water bath or Heat-block).
      5. After the heat shock, transfer the cells onto the ice and add 500uL of warm LB
      6. Place the tubes in the shaker (180 rpm) at 37°C for 1 hour.
      7. After the incubation, give a brief spin at 4000 rpm for 2-3 minutes and decant the supernatant of 400 ul. Mix the pellet with the remaining supernatant (~100uL)
      8. Split the mixture into 10uL and 90uL to spread onto different plates with appropriate antibiotics (or auxotrophic selection).
      9. Incubate the plates overnight at 37°C.
      10. After 12-16 hours of plating, colonies can be observed on the plates.

      Variations of the protocol

      Step 5:

      Adding 250 ul of media to the heat-shocked cells eliminates the spinning step, and one can directly spread the 10 % and 90 % volume on selective plates after 1-hour incubation.

      The plating of two different volumes helps in selecting well-spaced individual colonies for the downstream process.

      Storage

      The agar plates with bacterial colonies can be stored for a week @4°C. However, plates that contain antibiotics with less shelf-life may not be stored as without selection pressure, satellite colonies grow.

      Caution

      Do not leave the plates for more than the recommended time on incubator as overgrowth of colonies makes it challenging to pick individual ones, and it might produce satellite colonies (without plasmid).

      Comments Off on Heat shock transformation (E.coli) protocol